Stimulation of adrenoreceptors in mice induces an increase in cardiac mass unaffected by a reduction in afterload
Hypertrophy can result from pressure overload with increased wall stress, volume overload or catecholamine activation. Our study shows that the long term stimulation of α1- and/or β-adrenoreceptors induces a cardiac hypertrophy in adult male mice. ISO is a non-selective β-agonist acting primarily via the PKA pathway to up-regulate cyclic AMP and PE is a selective α1 agonist acting primarily via the PKC pathway through changes in Ca2+, phospholipids and diacylglycerol (Kobilka, 1993). We found the cardiac hypertrophy most pronounced when both α1- and β-adrenoreceptors are activated suggesting synergism when both PKA and PKC pathways are induced. ISO+PE-induced cardiac hypertrophy is more pronounced (P<0.05) than PE-induced cardiac hypertrophy throughout the time course studied whereas ISO-induced and ISO+PE-induced hypertrophy are not significantly different. A similar effect was seen in young piglets exposed to NE, an α1-agonist, a strong β1 agonist and weak activator of β2-adrenoreceptors, when compared to ISO or PE (Kolbilka, 1993; Buu et al., 1993; Cassidy et al., 1997). In this porcine study NE>ISO>PE on their measured indices of contractility and circulatory function. Thus, it appears that activation of the PKA pathway, rather than the PKC pathway is of more consequence in the in vivo generation of cardiac hypertrophy.
The extent of ISO-induced cardiac hypertrophy as measured by the heart to body weight ratio, did not decrease significantly within the time course. When both α- and β-adrenoreceptors are stimulated, as in the ISO+PE-treated mice, the increased cardiac mass is maintained throughout the time course. In a similar study, that also used osmotic minipumps to deliver the drugs, the HW/BW remained elevated in NE-treated versus saline-treated rats even when a reduced positive response to acutely given NE was detected (Laycock et al., 1995). The reduced acute response was attributed to a reduction in the number of β-adrenoreceptors in the NE-treated group. Downregulation of β1-adrenoreceptors was also shown in transgenic rats harbouring the mouse Ren-2d gene and in spontaneously hypertensive rats (Bohm et al., 1994; 1995). Although not proven, it is likely that a reduction in β-adrenoreceptors may have occurred in the ISO- and/or ISO+PE-treated animals despite the maintenance of an elevated HW/BW.
In our study, treatment with ISO, PE or ISO+PE increased mean arterial blood pressure to about the same level. Because the mice had been treated with the adrenergic agonists continuously for 7 days it is unlikely that any of the immediate acting pressure control mechanisms are operable (Guyton, 1992). We found that a reduction in mean arterial blood pressure by HL did not prevent the agonist-induced increase in cardiac mass. Similar results were found in ISO-induced cardiac hypertrophy (Golomb et al., 1994), in spontaneously hypertensive rat (Mitchell et al., 1996) or pressure overload rats (Susic et al., 1996). Thus, it appears that a reduction of blood pressure to a near normal level is insufficient to reduce ventricle weight in the presence of a second stimulus for hypertrophy, such as an excess of catecholamine. We and others have found that ISO-induced cardiac hypertrophy could be prevented by co-treatment with propranolol (β-adrenoreceptor antagonist) (Boluyt et al., 1995 and data not shown). We found that when the mini-pumps were removed a decrease in the HW/BW ratio to normal or approaching normal for all agonists occurred. These findings indicate that the maintenance of cardiac hypertrophy, although linked, is not solely dependent on afterload, but may be influenced by catecholamine excess. Further, these studies show that reversal of the hypertrophy can only be achieved by blockage or removal of agonist action suggesting that the catecholamine-induced hypertrophy results independent of agonist effects on blood pressure.
Concomitant with the agonist-induced increase in blood pressure was an increase in heart rate with the highest heart rate in ISO>ISO+PE>PE-treated mice. The increases in heart rate do not correlate with the increase in HW/BW or gene expression changes. The higher rate (P<0.01) in ISO-treated versus PE-treated mice was expected because of the known chronotropic effect of ISO acting on the β1-receptors in the myocardium and the increase in heart rate associated with constitutive expression of β2-adrenergic receptors in transgenic mice (Rockman et al., 1997; Milano et al., 1994). The heart rate in the ISO- versus ISO+PE-treated mice without HL was similar indicating no or undetectable effect of additional PE when ISO was infused. The heart rate increase in control mice treated with HL was expected because unopposed HL is known to cause an increase in heart rate in humans. Treatment with HL reduced the heart rate in PE and ISO+PE-treated mice to control levels, but was unable to reduce the heart rate to normal in ISO-treated mice. This is similar to the decrease in blood pressure, but not heart rate, found after captopril treatment of NE-induced hypertrophy in rats (Laycock et al., 1996). Our data suggest that unloading the heart is not sufficient to cause a reduction in heart rate if β-receptors alone are stimulated.
ANF and β-MHC induction
In agreement with most models of in vivo cardiac hypertrophy (reviewed in Schneider & Parker, 1993; van Bilsen & Chien, 1993; Pollak, 1995; Boluyt et al., 1995), our study shows induction of ANF by ISO, PE and ISO+PE, with the most pronounced induction by ISO+PE. Indeed, upregulation of ANF and β-MHC gene expression is a highly conserved event in ventricular hypertrophy both in vitro and in vivo in virtually all animals examined. We found ANF induction is closely correlated with cardiac mass increases, is maintained while agonists are present and further that ANF is decreased during reversal of cardiac hypertrophy. ANF with its vasodilator effect is thought to play a role in reducing haemodynamic load imposed on the heart and ANF knockout mice had a significantly increased blood pressure when compared to control littermates (John et al., 1995). However, we did not find lower arterial blood pressures in agonist-treated groups with the highest ANF levels suggesting that the higher ANF is insufficient to counteract the catecholamine action. Upregulation of β-MHC also correlated with cardiac mass increases, persists in the chronic phase of hypertrophy and is decreased after drug withdrawal. In physiological terms, the overexpression of the foetal isoforms may be a beneficial long-term adaptation to haemodynamic overload because β-MHC is thought to be bioenergetically more efficient than the adult isoform (Argentin et al., 1994). In our study, β-MHC expression is upregulated, but no change was detectable in α-MHC expression.
Neither ANF nor β-MHC expression decreased after an HL-mediated reduction of afterload in the PE- or ISO+PE-treated mice. Co-treatment of ISO-alone plus HL reduced the amount of β-MHC in comparison with PE-alone, but this level was still higher than in control mice. β-MHC expression was unaltered in pressure over-loaded rats treated with HL (Ardati & Nemer, 1993). These data suggest that upregulation of ANF or β-MHC levels is not closely tied to blood pressure, but rather is linked to agonist action, contractility and/or heart rate. In contrast, expression of α-MHC appears to be unaffected by either agonist action or the reduction in afterload caused by HL co-treatment. This is contradictory to findings in rats where α-MHC usually decreases in concert with an increase in β-MHC expression to result in a reversal of the α-MHC/β-MHC ratio (Schneider & Parker, 1993). We found a reduction in ANF and β-MHC, again without a change in α-MHC expression, only when the agonists were eliminated by removal of the minipumps. This result indicates that adjustments to a pre-treatment pattern of gene expression can only occur after agonist action on the heart itself is eliminated. These data suggest that control of α-MHC and β-MHC expression is different in mice compared to rats and that, whereas ANF and β-MHC are regulatable by agonists and blood pressure in mice, α-MHC expression is refractory.
Immediate early and muscle-specific transcription factor gene induction
Many studies have examined transient induction of transcription factors in the initiation of hypertrophy (Brand et al., 1993; Hannan & West, 1991; Schneider & Parker, 1993), but none of these studies examined their role in hypertrophy maintenance and regression. The present study shows that agonist-induced cardiac hypertrophy is accompanied by a continued high level of expression of c-fos, Egr-1, and, to a lower extent, c-jun. Thus, the data suggests that immediate early genes can be activated by both α- or β-adrenoreceptor, that activation of α- or β-agonists results in the highest levels in vivo and that the level of the immediate early genes is maintained by continuous infusion. The induction of their gene expression is well correlated with the extent of cardiac hypertrophy.
By comparing the pattern of expression after agonist stimulation, we observed that in adult cardiac hypertrophy, ISO, and to a lesser extent PE, induced c-fos and Egr-1, and that in ISO-PE-treatment the increase in c-jun, c-fos and Egr-1 expression was synergistic. The synergistic induction of Egr-1 and c-fos expression in our model suggests that both PKA and PKC pathways are involved in their induction. In other studies using aortic constriction to produce an acute pressure overload, c-fos induction was coupled to increases in cyclic AMP content and PKA activity in addition to a rise in PKC activity (Osaki et al., 1997) further implicating activation of both pathways in c-fos gene regulation in heart. In vitro binding of c-fos/c-jun heterodimers to ANF gene sequences has already been shown and it was suggested that the heterodimer plays a role in the regulation of ANF gene transcription in vivo (Rauscher et al., 1988; Rosenzweig et al., 1991; Barka et al., 1987). Correlation of the continued increase in ANF, parallel to increases in c-fos and c-jun expression, further implicates AP-1 activity in the maintenance of the high levels of ANF.
Egr-1 is a transcription factor that binds to a conserved GC-rich element found in such genes as ANF and α-MHC, and competes with Sp1 for DNA binding (Gashler & Sukhatme, 1995; Huang et al., 1997). We had previously found Egr-1 increased in every hypertrophied heart examined in our transgenic line expressing polyomavirus large T-antigen in heart (Holder et al., 1995). In the present study Egr-1 expression was increased primarily in ISO+PE and ISO-treated mice, with the smallest increase in PE-treated mice, at all times examined. Our results must be differentiated from the results of experiments performed by Brand et al. (1993) and Iwaki et al. (1990). Brand et al. (1993) treated isolated rat hearts for only 30 min to 1 h with ISO, PE or NE and found that PE and NE, but not ISO induced Egr-1 expression. The short time period of his experiments and the lack of a vascular component are likely to be significant differences affecting the outcome of the two studies. Iwaki et al. (1990) found Egr-1 upregulated in neonatal rat cardiomyocytes only after PE stimulation. However, significant differences exist between the gene expression in neonate versus adult cardiomyocytes and indicate that simple transfer of results from the neonatal cultures to adult heart can be problematic (Kitsis & Leinwand, 1992). Thus, differences may exist between the response of mouse and rat to adrenoreceptor stimulation and/or between cardiac hypertrophy induced by short stimulation (0.5–1 h) compared to cardiac hypertrophy induced by longer stimulation (3–14 days) or between neonatal cardiomyocyte culture in vitro and adult heart in vivo. Gupta et al. (1991) found Egr-1 regulated expression of α-MHC in neonatal rat cardiomyocytes. It is unclear if this activity is also present in adulthood as we found no change in α-MHC expression when Egr-1 levels were increased. This suggests either that Egr-1 does not control α-MHC expression in the adult or that other elements negate any Egr-1-mediated increase with no observable change in α-MHC expression as the end-result. Supporting the idea that Egr-1 does not control α-MHC expression was the finding of beating cardiomyocytes in differentiated embryonic stem cells deficient of Egr-1 and that adult mice lacking Egr-1 have apparent normal heart architecture (Lee et al., 1995).
The function of sustained expression of c-fos, c-jun and Egr-1 in heart pathology is unknown. It has been suggested that high levels of c-fos, c-jun and Egr-1 in terminally differentiated cells is correlated with growth arrest, progression and maintenance of the differentiation program (Gandarillas & Watt, 1995; Lord et al., 1993; Panterne et al., 1992; Liu et al., 1998; reviewed in Liebermann & Hoffman, 1998). In support of this idea, mice containing a fos-lacZ transgene show high levels of fos-driven lacZ in the most differentiated cells of the epidermis, hair follicle and epiphyseal plate with sustained expression preceding cell death (Smeyne et al., 1993). In another study high levels of c-fos induced growth inhibition of keratinocyte cell lines and increased their sensitivity to apoptosis (Mils et al., 1997). These studies suggest a correlation between high c-fos expression and bone and keratinocyte differentiation. However, overexpression of c-fos in transgenic mice led to dysregulation of bone growth and high expression in other tissues, including heart, led to no overt phenotype (Ruther et al., 1989; Bachiller and Ruther, 1990). Mice null for c-fos expression are viable, form a normal epidermis and did not form malignant papillomas when stimulated (Saez et al., 1995). Egr-1 deficient mice are also viable and display no obvious phenotype except for female infertility (Lee et al., 1995). Thus, the available transgenic and null expression results are in contrast to in vitro data. It may be that the other members of the Fos, Jun and Egr-1 families are able to overcome the defects in the transgene and null mutations. Adult cardiomyocytes have not been shown to proliferate to any great extent so it is unlikely that sustained expression of these genes is linked to increased proliferation in heart. It is more likely that, sustained expression of c-fos, c-jun and Egr-1 is involved in cardiomyocyte differentiation and/or apoptosis.
We show, for the first time, that the induction of Nkx-2.5 and GATA-4 can be produced by adrenereceptor stimulation with the highest increase seen when both α- and β-adrenoreceptors are stimulated. We suspect that GATA-4 expression can be equally initiated and maintained by either PKA or PKC activated mechanisms because GATA-4 expression was similar regardless of agonist stimulus and no synergism was detected if both pathways were activated simultaneously. High levels of Nkx-2.5 were found initially when both PKA and PKC pathways were activated in the ISO+PE-treated mice, but at later times Nkx2.5 was increased after 7 days of PE- or ISO-stimulation and this high level was maintained after 14 days. This indicates that co-stimulation by ISO and PE may serve to accelerate the time course for initiation of the high level of Nkx-2.5. The upregulation of GATA-4 and Nkx2.5 in our models of cardiac hypertrophy combined with its upregulation in aortic constriction and pressure overload, suggests that in increase in these genes may be a general feature of cardiac hypertrophy. They most likely play a role in initiating and maintaining the process of cardiac hypertrophy by activating cardiac promoters (Chen et al., 1996; Grepin et al., 1994; Durocher et al., 1996; Sepulveda et al., 1998).
Drug withdrawal after PE- or ISO-treatments is accompanied by a significant decrease in ANF, β-MHC, Egr-1, c-fos, c-jun and GATA-4 expression and a decrease in the HW/BW ratio to control levels. This implies that whereas continued expression of these genes is found when agonists are present once the heart has regressed to its normal weight maintenance of their expression is not required. Support for the notion that agonist stimulation is the important stimulant for their elevated expression is that when the agonist-treated animals were given HL and blood pressure was normalized the expression of these genes remained increased.
Heart regression was incomplete after ISO+PE stimulation as the HW/BW ratio remained elevated. In these hearts, ANF, β-MHC, Egr-1, c-jun, but not c-fos or GATA-4 remained elevated. This result suggests that Egr-1 and c-jun may be required for complete regression of heart weight to occur whereas continued expression of c-fos or GATA-4 is not necessary. Interestingly, GATA-4 and Nkx2.5 expression is very different after drug withdrawal. Nkx2.5 expression, although decreased, persists in the withdrawal after drug treatment regardless of the initiating stimulus and regardless of whether the HW/BW indicates complete or incomplete regression. In contrast, GATA-4 was decreased even when the HW/BW indicated incomplete regression. The implication is that regression might be a dynamic process that requires the expression of a subset of transcription factors. Further, the data also suggests that although HW/BW ratios and determination of ANF or β-MHC levels might indicate that complete regression has occurred considerable molecular changes might still be underway that require specific transcription factors.
Recognition of the central role that transcription regulatory genes play in gene expression led us to search for specific gene expression changes in the hypertrophying heart. How the different transcription factors involved in cardiogenesis operate in the entire regulatory cascade of cardiac hypertrophy has yet to be fully delineated. We found that Egr-1, c-fos, c-jun, Nkx2.5 and GATA-4 are upregulated in chronic-induced cardiac hypertrophy, that the induction is not transient, but persists through the time course. We found that Egr-1, c-fos, c-jun, Nkx2.5 and GATA-4 expression does not decrease when the blood pressure is reduced suggesting that direct drug action of adrenoreceptor agonists on cardiomyocyte is more important in inducing gene expression than afterload reduction. Regression after drug withdrawal appears to be an active process that requires a subset of specific transcription factors. To investigate the role of c-fos and Egr-1 in response to hypertrophic stimuli and to dissect the mechanism of cardiac hypertrophy, we are extending our study to include NGFI-A−/− (null mutant mice for Egr-1 gene) (Lee et al., 1995) and/or c-fos−/− (null mutant mice for c-fos gene) (Wang et al., 1992) as subjects for these in vivo models.